Ribonuclease H (RNase H) is one of a family of enzymes termed nucleases, which act to hydrolyse nucleic acids. RNase H is unique among nucleases in that it selectively degrades the RNA component of an RNA/DNA duplex molecule, a double-strand nucleic acid comprised of one strand of ribonucleic acid (RNA) bound to a complementary strand of deoxyribonucleic acid (DNA) via Watson-Crick base pairing. Ribonuclease H enzymes are ubiquitous, found in virtually all organisms, as well in several types of virus1.
Ribonuclease H performs critical functions in the replication of several human pathogenic viruses, including retroviruses such as the human immunodeficiency virus (HIV) types 1 and 2, and the human T-cell leukaemia viruses (HTLV) types 1 and 2. In addition, ribonuclease H is essential for the replication of the human hepadnavirus, hepatitis B virus (HBV).
Retroviral Ribonuclease H. Retroviruses carry their genetic information as RNA, but must replicate through a double-strand DNA intermediate. Thus, following recognition and entry into a susceptible cell, the retroviral genomic RNA must be converted into viral DNA. Multiple steps are involved in this crucial step of replication, each of which is catalyzed by the retroviral enzyme reverse transcriptase (RT). This enzyme is therefore multifunctional, and possesses three enzymatic activities, RNA-dependent DNA polymerase activity (RDDP), DNA-dependent DNA polymerase activity (DDDP), and ribonuclease H activity (RNase H).
Several retroviruses are human pathogens. These include the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), and the human T-cell leukemia viruses types 1 and 2 (HTLV-1 and HTLV-2). Of these, HIV-1 is by far the most serious pathogen. HIV-1 infection leads to AIDS, an incurable and inevitably fatal disease. Since identification of the virus in the early 1980's, it is estimated that more than 58 million individuals have been infected with HIV-1, and of these nearly 25 million have died of AIDS. HIV-1 infection remains one of the most serious infectious disease problems worldwide.
A variety of biological agents are currently in use for the treatment of HIV-1 infections. HIV-1 RT has been, and remains, an important target for antiviral development. Many inhibitors of HIV-1 RT have been discovered, including nucleoside reverse transcriptase inhibitors (NRTI) such as 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxy-3′-thiacytidine (3TC) and nonnucleoside reverse transcriptase inhibitors (NNRTI) such as nevirapine, delavirdine and efavirenz (for a recent review see reference 2). However, virtually all inhibitors of HIV-1 RT are directed against the RDDP and/or DDDP activity of RT. Very few inhibitors of the ribonuclease H activity of HIV-1 (and HIV-2) reverse transcriptase have been described, and none are in clinical use.
Although current therapeutics are initially very effective at controlling the course of HIV spread in an infected individual, thereby improving the quality of life and longevity of HIV-infected patients, prolonged therapy inevitably leads to viral resistance to these drugs. Resistance to RT inhibitors correlates with mutations in RT, and resistance to protease inhibitors correlates with mutations in the HIV protease. Clinical appearance of drug-resistant HIV imparts an unfavorable prognosis. In addition, the transmission of drug-resistant HIV variants from an infected treated individual to a previously naïve individual is becoming a serious problem. Drug therapies for use by these newly infected patients are restricted because of the infection by drug-resistant virus. There is therefore an urgent need to identify new inhibitors of HIV replication, especially inhibitors that act on new viral targets, not presently targeted by current chemotherapies. These new targets include the ribonuclease H activity associated with the viral reverse transcriptase. Current assays for this enzyme activity are cumbersome and time-consuming, and unsuitable for high-throughput screening of the large chemical libraries available to major pharmaceutical companies. In order to identify inhibitors of this viral enzyme, more appropriate assays are needed, particularly assays suitable for high-throughput screening of large libraries of chemical compounds.
Hepadnaviral Ribonuclease H. Human hepatitis B virus (HBV) is a major worldwide health threat and is responsible for the majority of the 1 to 2 million deaths annually from hepatitis. HBV is a member of the hepadnavirus family. Hepadnaviruses are small enveloped DNA viruses that replicate through an RNA intermediate. This replication mechanism therefore requires reverse transcription, to convert the RNA intermediate into viral DNA, a process carried out by the hepadnaviral P protein. As is the case with retroviral reverse transcriptases, hepadnaviral P protein must be multifunctional to carry out reverse transcription. Thus, the protein possesses RNA-directed DNA polymerase and DNA-directed DNA polymerase activities, and ribonuclease H activity.
There are very few treatments available for HBV infection. These include interferon therapy or liver transplantation, both of which are expensive and at best only partially successful. Recently, the nucleoside analog 3TC has been approved for treatment of chronic infection and transplant patients. This nucleoside is directed against the DNA polymerase activity of the HBV DNA polymerase (hepadnaviral P protein). Additional therapies need to be developed. The hepadnaviral P protein-associated ribonuclease H provides a target for this development. Again, as is the case for retroviral RT RNase H, assays for this enzyme activity are cumbersome and time-consuming, and unsuitable for high-throughput screening of the large chemical libraries available in major pharmaceutical companies. In order to identify inhibitors of this viral enzyme, improved assays are needed, e.g. suitable for high-throughput screening of large libraries of chemical compounds.
Assays for Ribonuclease H Activity. Several types of assays for ribonuclease H have been described. For the large part, these assays involve the use of radiolabeled RNA/DNA duplex substrates. RNase H degradation of the radiolabeled RNA strand releases small RNA fragments which are then measured and/or visualized by various means, including liquid scintillation counting, autoradiography, etc.
1. Radioactivity release assay3,4. In this assay, the RNA/DNA hybrid duplex substrate is prepared such that the RNA is uniformly radiolabeled throughout its length either by incorporation of [3H]-NTPs or [α-32P]-NTPs during in vitro synthesis of the RNA transcript used in the preparation of the RNA/DNA duplex substrate. Incubation of the radiolabeled RNA/DNA hybrid duplex substrate with RNase H results in degradation of the RNA into small radiolabeled RNA fragments (generally less than or equal to 3 nucleotides in size). The reaction is stopped and any unreacted large radiolabeled RNA/DNA hybrid duplex substrate is then precipitated by the addition of acid. After high-speed centrifugation, aliquots of the supernatant are counted by liquid scintillation spectrometry. The amount of radioactivity in the supernatant is directly related to the extent of RNase H degradation of the radiolabeled RNA/DNA hybrid duplex substrate. In a variation of this method4, the synthetic radiolabeled RNA/DNA hybrid duplex substrate is immobilized on nitrocellulose filters by UV irradiation. Treatment of the filters with RNase H degrades the RNA, releasing small radiolabeled fragments into the solution. RNase H activity is then measured by determining the increase in solution radioactivity, and/or by the decrease in filter-bound radioactivity, using liquid scintillation spectrometry.
2. Renaturation gel assay5-7. Ribonuclease H enzymes, especially those of prokaryotic origin, are readily renatured following denaturation with agents such as sodium dodecyl sulfate (SDS). In the renaturation gel assay, the RNase H is electrophoresed in an acrylamide gel in which an RNA/DNA hybrid duplex (with the RNA strand radiolabeled, usually by 32P) has been embedded by copolymerization. Following electrophoresis, the RNase H is renatured by soaking the gel in various buffers appropriate for renaturation, and then placed in a buffer that allows initiation of RNase H activity (usually by the presence of divalent metal cations such as Mg2+ or Mn2+). After an appropriate period (generally 15–20 hours or longer), the gel is fixed in an acidic solution, dried, and the distribution of radioactivity visualized by autoradiography. Areas of RNase H activity will appear as a light-to-white band on a dark background. This “negative” detection is difficult to quantitate accurately, thus the gel renaturation method provides only qualitative assessment of RNase H activity. A non-radioactive version of this assay has been described7 in which the RNA is synthesized with the fluorescent nucleotide BODIPY-TR-14-UTP. Following electrophoresis, renaturation and RNase H activity, the loss of fluorescence in the area of Rnase H is visualized by a fluorescence scanner. As with the radioactive version, this fluorescence assay is qualitative only.
3. Gel electrophoretic assay8,9. In this assay, the RNA is radiolabeled with 32P, either by synthesis using [α-32P]-NTPs, or more commonly by labeling of the 5′-end of a synthetic RNA using [γ-32P]-ATP and bacteriophage T4 polynucleotide kinase. The [32P]-labeled RNA is hybridized to a complementary DNA to form the RNA/DNA hybrid duplex substrate. Addition of RNase H degrades the RNA strand. The extent of degradation, and in many cases the degradation products, are visualized by removing aliquots of the reaction mixture at various times, separating the reaction products by electrophoresis on denaturing sequencing gels, followed by autoradiography. The extent of RNase H activity can be determined quantitatively by densitometric analysis of the time-dependent disappearance of the full-length RNA substrate and/or the appearance of the smaller degradation products. Variations of the gel electrophoretic assay include the use of capillary gel electrophoresis coupled with UV detection to identify degradation products10, or visualization of unlabeled RNA degradation products after polyacrylamide gel electrophoresis using a general nucleic acid stain such as Stains-All. The latter method suffers from lack of sensitivity, especially for small RNA degradation products.
4. Other assay methods. A non-radioactive solution-phase assay has been described11 that uses a 5′-biotin-RNA strand duplexed with a 5′-digoxigenin-DNA strand. Treatment with RNase H cleaves the biotinylated RNA from the RNA/DNA hybrid duplex substrate. Aliquots are removed at various times of reaction, and any remaining uncleaved substrate is captured onto streptavidin-coated 96-well microtiter plates followed by detection with an alkaline phosphatase-labeled anti-digoxin antibody.
All of these methods provide only discontinuous measurements of RNase H activity, and involve sample handling at fixed time points. Kinetic measurements therefore require that aliquots be removed from a reaction pool, and analyzed individually, a time-consuming process which can limit the precision of the assay. In addition, many of the methods require one or more additional steps, such as electrophoretic resolution of the degraded RNA followed by autoradiography, ELISA detection of residual unreacted modified RNA, etc. All of these additional steps add to the time needed to complete the assay. Many of these additional steps, such as electrophoretic separation of reaction products, are not amenable to high-throughput analysis.
Nucleases, which are enzymes which hydrolyze/cleave nucleic acids, are differentiated by the identity of the substrates on which they act. A variety of assays are available to measure different types of nuclease activity, including gel electrophoresis (similar to that described above), thin-layer chromatography12, capture and elution of products from ion-exchange filters13, etc. As with the other methods described above, none of these assays described to date are suitable for real-time kinetic measurements, and all involve one or more additional steps following the nuclease cleavage of the substrate nucleic acids.
There therefore is a need for an improved RNase H assay.